Transglutaminase 2-Catalyzed Intramolecular Cross-Linking of

Dec 17, 2015 - Polymerization of OPN by intermolecular cross-linking has mostly been studied using relatively ... Collectively, these data suggest tha...
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Transglutaminase 2‑Catalyzed Intramolecular Cross-Linking of Osteopontin Brian Christensen,† Elias D. Zachariae,† Carsten Scavenius,† Søren Kløverpris,† Claus Oxvig,† Steen V. Petersen,‡ Jan J. Enghild,†,§ and Esben S. Sørensen*,†,§ †

Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark § Interdisciplinary Nanoscience Center, Aarhus University, 8000 Aarhus C, Denmark ‡

S Supporting Information *

ABSTRACT: Osteopontin (OPN) is a multifunctional integrin-binding protein present in several tissues and body fluids. OPN is a substrate for the enzyme transglutaminase 2 (TG2), which catalyzes inter- and intramolecular cross-linking affecting the biological activity of the protein. Polymerization of OPN by intermolecular cross-linking has mostly been studied using relatively high TG2 concentrations, whereas the effect of lower concentrations of TG2 has remained unexplored. Here we show that TG2 at physiologically relevant concentrations predominantly catalyzes the formation of intramolecular cross-links in OPN. By site-directed mutagenesis and mass spectrometry, we demonstrate that Gln42 and Gln193 serve as the primary amine acceptor sites for isopeptide bond formation. We find that Gln42 predominantly is linked to Lys4 and that Gln193 participates in a cross-link with Lys154, Lys157, or Lys231. The formation of specific isopeptide bonds was not dependent on OPN phosphorylation, and similar patterns of cross-linking were observed in human and mouse OPN. Furthermore, we find that OPN purified from human urine contains the Lys154−Gln193 isopeptide bond, indicating that intramolecular cross-linking of OPN occurs in vivo. Collectively, these data suggest that specific intramolecular cross-linking in the N- and C-terminal parts of OPN is most likely the dominant step in TG2-catalyzed modification of OPN.

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to changes in the structure, solubility, and function of the implicated proteins.17 TG2 has been shown to cross-link OPN into high-molecular weight polymeric aggregates both in vitro and in vivo.11,12,15 In vitro studies have shown improved cell binding properties of TG2-polymerized OPN leading to enhanced cell adhesion and migration.10,12 TG2-catalyzed polymerization of OPN can induce formation of de novo binding sites for integrins, as the α9β1 integrin binds polymeric OPN in a manner independent of the SVVYGLR motif.13 It has been shown that polymeric OPN induces chemotactic recruitment of neutrophils in vivo14 and enhances opsonization.18 Recently, we identified Gln34, Gln42, Gln193, and Gln248 as the major TG2 reactive glutamines in human OPN and showed that reactive Lys residues are mainly located in the C-terminal part of the protein.19 Previous studies of TG2-catalyzed cross-linking of OPN have mainly focused on the formation of high-molecular weight aggregates, and polymerization of OPN has been demonstrated using relatively high TG2:OPN ratios [up to 1:1 (w/w)].10−14 Intramolecular cross-linking of OPN has received less attention, though it has

steopontin (OPN) is a highly acidic multifunctional protein implicated in a wide range of biological processes, including bone remodeling, inhibition of ectopic calcification, tumorigenesis, and immune modulation.1 OPN is a member of the SIBLING (small integrin-binding ligand N-linked glycoprotein) protein family2,3 and is expressed by a wide variety of cell types. OPN is present in several tissues like bone, mammary gland, brain, and kidney and is secreted into body fluids such as blood, milk, and urine.1,4,5 OPN−integrin interaction regulates many aspects of cell behavior, including cell adhesion, migration, and immune modulation.1,6 Different integrin receptors bind OPN through either the conserved RGD sequence or a cryptic SVVYGLR motif exposed upon proteolytic cleavage.1,6 OPN is an intrinsically disordered protein extensively modified by phosphorylation, O-glycosylation, and proteolytic processing by matrix metalloproteinases, plasmin and especially thrombin, which influences the ability of OPN to mediate RGD-dependent cell adhesion, stimulate bone resorption, and regulate mineralization.2,6−9 OPN is a known substrate for transglutaminase 2 (TG2), also known as tissue transglutaminase.10−15 TG2 is a widely distributed intra- and extracellular calcium-dependent enzyme that catalyzes the formation of isopeptide bonds between specific lysyl and glutamyl residues in substrate proteins.16 Isopeptide bonds can be formed both as inter- and intramolecular cross-links and lead © XXXX American Chemical Society

Received: October 22, 2015 Revised: December 17, 2015

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DOI: 10.1021/acs.biochem.5b01153 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry been observed in some studies.10,14,20 The concentration of TG2 in homogenates of human cerebral cortex and in lysates of human HeLa cells has been reported to be in the range of ∼1− 1.3 ng/mL.21 The TG2 concentration in human urine has been estimated to be 82 ng/mL,22 which compared to the urinary concentration of OPN of 4 μg/mL23 gives a TG2:OPN ratio in human urine of approximately 1:50 (w/w). In this study, we investigate the cross-linking of OPN at TG2:OPN ratios from 1:100 to 1:20 (w/w) resembling in vivo ratios.21−25 Under these conditions, we show that TG2 catalyzes the formation of intramolecular cross-links in OPN, whereas no polymerization is observed. By site-directed mutagenesis and mass spectrometric analyses, Gln42 and Gln193 were demonstrated to serve as the primary amine acceptor sites in the isopeptide bonds. Gln42 was shown to be predominantly linked to Lys4, and Gln193 was demonstrated to participate in a cross-link with Lys154, Lys157, or Lys231. We, furthermore, show that intramolecularly cross-linked OPN exists in vivo in human urine.

dialyzed against 25 mM ammonium bicarbonate, and lyophilized. Mouse OPN from medium conditioned by 275-3-2 murine ras-transformed fibroblasts and OPN from human urine and milk were purified as previously described.5,7,27 Cross-Linking Reactions. Recombinant human OPN was incubated with TG2 using an enzyme:substrate ratio of 1:100, 1:75, 1:50, 1:30, or 1:20 (w/w) in 40 mM Tris-HCl (pH 8.3), 140 mM NaCl, 10 mM CaCl2, and 5 mM dithioerythritol at 37 °C for 3 h. In another experiment, recombinant human OPN and human milk OPN were incubated with TG2 using an enzyme:substrate ratio of 1:5 (w/w) for 4 and 16 h. All reactions were quenched by the addition of ethylenediaminetetraacetic acid to a final concentration of 50 mM, and the degree of cross-linking was analyzed by 18% SDS−PAGE followed by Coomassie Brilliant Blue staining. OPN samples incubated with TG2 at an enzyme:substrate ratio of 1:15 (w/w) for 3 h at 37 °C were dialyzed against 50 mM ammonium bicarbonate and digested with thrombin (30 milliunits/μg of OPN) at 37 °C for 1 h and subsequently analyzed by 18% SDS−PAGE and linear MALDI-MS. The Nand C-terminal fragments were separated by reverse phase high-performance liquid chromatography (RP-HPLC) as described previously,9 and the N-terminal fragment was digested with endoproteinase Glu-C as described below. Mouse OPN and TG2 were incubated at a 10:1 ratio (w/w) for 3 h at 37 °C, and the degree of cross-linking was analyzed by Western blotting using monoclonal anti-OPN antibody 2A1 (1.5 μg/mL).28 Identification of Residues Involved in Intramolecular Cross-Linking of OPN. Individual bands of recombinant human OPN after incubation with TG2 were excised from Coomassie Brilliant Blue-stained SDS−PAGE gels and destained in a 50 mM ammonium bicarbonate/50% acetonitrile mixture, followed by in-gel digestion with trypsin in 50 mM ammonium bicarbonate overnight at 37 °C. The resulting tryptic peptides were desalted and concentrated on a Zip-tip column containing C 18 reversed phase (RP) material (Millipore) and analyzed by MALDI-MS. TG2-treated and untreated OPN were also digested with trypsin [1:50 (w/w)] at 37 °C for 6 h, and the resulting peptides were separated by RPHPLC on a C2/C18 PC 2.1/10 column connected to a GE Healthcare SMART system. Separation was conducted in 0.1% trifluoroacetic acid (buffer A) and eluted with a gradient of 60% acetonitrile in 0.1% trifluoroacetic acid (buffer B) developed over 54 min (from 0 to 9 min, 0% buffer B; from 9 to 49 min, 0 to 50% buffer B; 49 to 54 min, 50 to 100% buffer B) at a flow rate of 0.15 mL/min. Eluted peptides were detected by measuring the absorbance at 214 nm and analyzed by MALDIMS. For further analysis, wild-type (wt) OPN and the Q36A and Q42A OPN mutants were incubated with or without TG2. The proteins and the N-terminal fragments resulting from thrombin cleavage were subsequently digested with endoproteinase GluC [1:25 (w/w)] in 50 mM ammonium bicarbonate at 37 °C for 3 h. The resulting peptides were separated by RP-HPLC as described above. Fractions containing cross-linked peptides were further digested with 50 ng of trypsin or endoproteinase Asp-N in 50 mM ammonium bicarbonate at 37 °C for 5 h. TG2-treated and untreated mouse OPN were digested with trypsin using an enzyme:substrate ratio of 1:50 (w/w) at 37 °C for 16 h. The resulting peptides were concentrated on Zip-tip columns and analyzed by MALDI-MS.

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EXPERIMENTAL PROCEDURES Materials. Sequencing grade modified trypsin was from Promega. Guinea pig TG2, endoproteinase Glu-C, Asp-N, and human thrombin were from Sigma. The mono S HR 10/10 and C2/C18 PC 2.1/10 columns, pGEX6P2 plasmid, and glutathione-Sepharose 4B were from GE Healthcare. PreScission Protease was from Fisher Scientific. Cloning, Expression, and Purification of OPN Variants. A construct containing residues 1−298 of human OPN (without the signal peptide) and an N-terminal His tag26 was amplified by polymerase chain reaction using the 5′-primer cagaggatcccatcaccatcacc and the 3′-primer cgacctcgagctactaattgacc and then cloned into the BamHI and XhoI sites within the multiple cloning site of the pGEX6P2 vector. This plasmid was used as template for site-directed mutagenesis reactions using a QuikChange site-directed mutagenesis kit (Stratagene). The following mutations were introduced using appropriate mutagenesis primers: Q36A, Q42A, and Q193A. All constructs were propagated in Escherichia coli DH5α cells and verified by sequence analysis. Plasmid DNA for transformation was prepared using a NucleoSpin Plasmid QuickPure Kit (Macherey-Nagel). Cells of E. coli strain BL-21cc were transformed with the expression vector containing OPN using heat shock and grown on ampicillin-containing plates. Individual colonies were picked and propagated overnight in 50 mL of lysogeny broth [1% tryptone, 0.5% yeast extract, 1% glycose, and 1% NaCl (pH 7.0)] with 100 μg/mL ampicillin at 37 °C. The volume was increased to 1000 mL and the culture incubated at 37 °C until the OD600 reached 0.6, at which time isopropyl 1-thio-β-Dgalactopyranoside was added to a final concentration of 0.1 mM. Cultures were grown for several more hours until the OD600 reached 1.2−1.5, at which point the cells were collected and sonicated. Glutathione S-transferase fusion proteins were affinity-purified with glutathione-Sepharose 4B resin, eluted with free glutathione, and then cleaved off from glutathione Stransferase with PreScission protease after elution from the resin. The purity of the product was confirmed by 18% SDS− PAGE. The recombinant OPN was further purified using a 1 mL metal-chelate affinity column (Qiagen) charged with nickel ions. Bound protein was eluted with 250 mM imidazole in PBS. The fractions containing OPN were identified by SDS−PAGE, B

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Figure 1. TG2-mediated intramolecular cross-linking of OPN. (A) Human recombinant OPN and (B) human milk OPN were incubated with TG2 at different enzyme:substrate ratios for 3 h. The reaction products were separated by 18% SDS−PAGE and stained with Coomassie Brilliant Blue. The enzyme:substrate ratios (w/w) are indicated above the gel. Non-cross-linked OPN and intramolecularly cross-linked OPNi are indicated with arrows.

Figure 2. Thrombin cleavage of intramolecularly cross-linked OPN. (A) OPN was incubated without (lanes 1 and 3) or with (lanes 2 and 4) TG2 in a 15:1 ratio (w/w) and subsequently cleaved with thrombin (lanes 3 and 4). The reaction products were separated by 18% SDS−PAGE and stained with Coomassie Brilliant Blue. (B) MALDI-MS of thrombin-cleaved OPN and OPNi. The calculated molecular masses of the N- and C-terminal parts of OPN are 18238.9 and 16843.1 Da, respectively.

Identification of Cross-Linked OPN in Human Urine. OPN purified from human urine was digested with trypsin using an enzyme:substrate ratio of 1:50 (w/w) in 50 mM ammonium bicarbonate at 37 °C for 16 h. The resulting peptides were desalted on C18 stage tips (Proxeon, Thermo Scientific) and subjected to nano-liquid chromatography− tandem mass spectrometry (LC−MS/MS) analysis. Mass Spectrometry. MALDI-MS was performed using a Bruker Autoflex III instrument operated in linear mode and calibrated in the mass range of 5000−17500 Da using Protein Calibration Standard I (Bruker Daltonics). MS and MS/MS

were also performed on a Q-TOF Ultima MALDI mass spectrometer (Micromass) calibrated over the m/z range of 50−3000 using a polyethylene glycol mixture. External calibration of each MS spectrum was performed using Glufibrinopeptide B (m/z 1570.6774). The theoretical peptide masses were provided by the GPMAW software (Lighthouse Data). Nano-LC−MS/MS analyses were performed on an EASYnLC II system (Thermo Scientific) connected to a TripleTOF 5600+ mass spectrometer (AB Sciex) equipped with a NanoSpray III source (AB Sciex) operated under the control C

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Biochemistry of Analyst TF version 1.5.1. The tryptic peptides were dissolved in 0.1% formic acid, injected, trapped, and desalted isocratically using a trap column containing ReproSil-Pur C18-AQ 3 μm resin (Dr. Marisch GmbH) [2 cm × 100 μm (inside diameter)]. The peptides were eluted from the trap column and separated on a 15 cm analytical column (75 μm inside diameter) packed in house in a pulled emitter with RP ReproSil-Pur C18-AQ 3 μm resin (Dr. Marisch GmbH) at a rate of 250 nL/min using a 30 min gradient from 5 to 35% phase B (90% acetonitrile in 0.1% formic acid). Detection of in vivo-generated intramolecularly cross-linked OPN from urine was performed by multiple-reaction monitoring at high resolution (MRM-HR). A MS scan was acquired (m/z 350−1250, 0.25 s) followed by product ion scans for m/z 762.7 and 592.3. The product ion scans were 100 ms in the mass range of m/z 200−1800. Identification of the intramolecularly cross-linked peptides was based on a manual inspection of MS/MS spectra with a chromatographic retention time matching that of a control sample (TG2-treated OPN). The collected MS files were converted to Mascot generic format using the AB SCIEX MS Data Converter beta 1.1 (AB SCIEX) and the “proteinpilot Mascot generic format” parameters. The generated peak lists were searched against the swiss-prot database using an in-house Mascot search engine (matrix science). The following search parameters were used: Homo sapiens, trypsin, two missed cleavages, oxidation (Met) variable modification, and 2+, 3+, and 4+ charges. Peptide and MS/MS tolerances were set to 10 ppm and 0.2 Da, respectively. All spectra were inspected manually.

Figure 3. MALDI-MS analysis of trypsin-digested OPN and OPNi. (A) MALDI-MS of tryptic peptides from in-gel digests of OPN and OPNi (Figure 2A, lanes 1 and 2). (B) MS/MS analysis of the parental fragment ion at m/z 2286.12 observed in OPNi in which peptides 153 SKSK156 and 188AIPVAQDLNAPSDWDSR204 were shown to be cross-linked.

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RESULTS Intramolecular Cross-Linking of OPN. Recombinant human OPN was incubated with TG2 at different enzyme:substrate ratios, and the reaction products were analyzed by SDS− PAGE. A band migrating further in the gel than non-crosslinked OPN was detected at an enzyme:substrate ratio of 1:100 (w/w) (Figure 1A). This indicates that TG2 introduced intramolecular isopeptide bonds in OPN creating a more compact form of the protein. In the following, the intramolecularly cross-linked OPN form will be termed OPNi. The band representing OPNi became more intense with increasing concentrations of TG2 (Figure 1A). The highest intensity of the OPNi band was observed at an enzyme:substrate ratio of 1:20 (w/w), and the intensity of both OPN bands at the highest TG2 concentrations did not suggest noticeable formation of higher-molecular weight OPN polymers (Figure 1A). The TG2-mediated formation of OPNi was not dependent on the modification status of OPN, as highly phosphorylated and glycosylated human milk OPN also formed intramolecular cross-links (Figure 1B). Using an enzyme:substrate ratio of 1:5 (w/w), a faint band also appeared at the interface between the stacking and resolving gel (Figure S1), indicating OPN polymerization, though most OPN is still intramolecularly cross-linked even at this higher TG2 concentration. OPN was incubated with and without TG2, followed by thrombin cleavage that separates the N- and C-terminal parts of OPN by cleavage at the Arg152−Ser153 bond.9 Thrombin cleavage of both non-cross-linked OPN and OPNi resulted in two bands representing the N- and C-terminal regions, respectively, and further shows that no cross-links were formed between these two regions (Figure 2A, lanes 3 and 4). However, TG2 treatment induced faster migration of both fragments, indicating the presence of isopeptide bonds within

both regions. The thrombin-cleaved fragments were further analyzed by MALDI-MS to determine the number of TG2induced cross-links. The calculated average molecular masses of the unmodified N- and C-terminal regions are 18238.9 and 16843.1 Da, respectively. The masses of both fragments were observed to be reduced by ∼15 Da after TG2 treatment (Figure 2B), indicating the presence of a single intramolecular cross-link in both the N-terminal and C-terminal regions, as the formation of an isopeptide bond between Lys and Gln residues (transamidation) results in a mass loss of 17 Da. Identification of Residues Involved in TG2-Mediated Intramolecular Cross-Linking of OPN. The bands representing OPN and OPNi (Figure 2A, lanes 1 and 2) were excised, in-gel-digested with trypsin, and analyzed by MALDIMS. A peptide with an m/z value at 1854.88 corresponding to peptide 188AIPVAQDLNAPSDWDSR204 was not observed after TG2 treatment (Figure 3A). Conversely, a peptide with an m/z value at 2286.12 corresponding to a dipeptide formed by cross-linking of 153SKSK156 with 188AIPVAQDLNAPSDWDSR204 (m/zcalculated 2286.13) was observed after TG2 treatment of OPN. The identity of this cross-linked peptide was further established by MS/MS analysis (Figure 3B), which confirmed a cross-link between Lys154 and Gln193. The crosslinked peptides from the N-terminal region were not identified by this approach. To further characterize the residues that are intramolecularly cross-linked by TG2, OPN and OPNi were digested with trypsin and the resulting peptides were separated by RP-HPLC D

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Figure 4. Analysis of trypsin-digested OPN and OPNi. (A) Peptides from trypsin digests of OPN and OPNi were separated by RP-HPLC and eluted with a gradient of 60% acetonitrile in 0.1% trifluoroacetic acid (---). The peptides were detected in the effluent by measuring the absorbance at 214 nm (). (B) Characterization of peptides from fractions A−D in panel A. In some cases, the samples were further digested with endoproteinase Asp-N. Monoisotopic or average (†) molecular masses (MH+) were measured by MALDI-MS. Difference denotes the mass difference between measured and expected masses calculated using the GPMAW software. The type of modification corresponding to the mass difference is a cross-link (cl) and pyroglutamic acid formed from N-terminal glutamines (pGlu).

Figure 5. Analysis of the N-terminal part of OPN and OPNi digested with Glu-C. (A) The N-terminal parts of OPN and OPNi resulting from thrombin cleavage were digested with Glu-C. The peptides were separated by RP-HPLC and eluted with a gradient of 60% acetonitrile in 0.1% trifluoroacetic acid (---). The peptides were detected in the effluent by measuring the absorbance at 214 nm (). (B) Characterization of peptides from fractions 1−4 in panel A. In some cases, the samples were further digested with trypsin or endoproteinase Asp-N. Monoisotopic or average (†) molecular masses (MH+) were measured by MALDI-MS. Mass difference is between measured (not shown) and expected masses calculated using the GPMAW software. The type of modification corresponding to the mass difference is a cross-link (cl) and hydration due to proteolytic cleavage (H2O). The residues identified to be cross-linked are listed when possible.

most likely partner because of the formation of pyroglutamic acid from Gln36 (Figure 4B). To further investigate the residues involved in the N-terminal part, OPN and OPNi were digested with thrombin and the N-terminal part was purified by RPHPLC (data not shown) and further digested with endoproteinase Glu-C. The resulting peptides were separated by RP-HPLC (Figure 5). The most significant differences upon comparison of the elution profiles were the strong reduction in the signal intensity of fractions containing the peptides His6I1PVKQADSGSSEE13 and K14QLYNKYPDAVATWLNPDPSQKQNLLAPQNAVSSEE49 after TG2 treatment, consistent with the formation of a cross-link between Lys4 and Gln36 or Gln42. The two minor fractions 3 and 4 appearing in the OPNi

(Figure 4). The most significant differences between the elution profiles were the disappearance of the peptide 188AIPVAQDLNAPSDWDSR204 in OPNi and the appearance of three fractions in OPNi containing cross-linked peptides identified by a mass loss of 17 Da. MS analysis of these fractions showed that Gln193 can be cross-linked to Lys154, Lys157, or Lys231 in the C-terminal region of OPN. As estimated by the absorption at 214 nm (Figure 4A), the isopeptide bond between Lys154 and Gln193 is the major cross-link in the C-terminal part. The two N-terminal peptides, His6-Ile1−Lys14 and Gln36−Lys61, were also observed to be cross-linked by TG2. Subdigestion of this fraction with endoproteinase Asp-N showed the presence of a cross-link between Lys4 and Gln36 or Gln42, with Gln42 as the E

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Figure 6. RP-HPLC of Glu-C-digested wt OPN and Q36A and Q42A mutants. (A) wt OPN and Q36A and Q42A mutants were cross-linked with TG2 and digested with Glu-C. The peptides were separated by RP-HPLC and eluted with a gradient of 60% acetonitrile in 0.1% trifluoroacetic acid (---). The peptides were detected in the effluent by measuring the absorbance at 214 nm (). The peptides present in fractions A−C were identified by MALDI-MS. (B) Characterization of peptides in Q36A fractions 1 and 2 in panel A. In some cases, the samples were further digested with trypsin or endoproteinase Asp-N. Monoisotopic or average (†) molecular masses (MH+) were measured by MALDI-MS. Mass difference is between measured and expected masses calculated using the GPMAW software. The type of modification corresponding to the mass difference is a cross-link (cl) and hydration due to proteolytic cleavage (H2O). The residues identified to be cross-linked are listed when possible.

Figure 7. Schematic representation of intramolecularly cross-linked OPN. The most dominant cross-links identified in this study are indicated with black lines, and the Gln and Lys residues are shown in boldface. Minor cross-links are indicated with gray dashed lines. All Gln residues in OPN and the thrombin cleavage site are indicated. The integrin-binding RGD and SVVYGLR motifs are shown in italics.

F

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further verifying a cross-link involving Gln193. A schematic presentation of the residues involved in the cross-linking of OPNi is shown in Figure 7. Gln42 and Gln193 Are Specific TG2 Targets. OPN exhibited faster migration in SDS−PAGE after TG2 crosslinking (Figure 1) probably because of structural compaction of the protein. Gln 42 and Gln 193 were identified as the participating glutamines in intramolecular TG2 cross-linking of OPNi. Their influence on the migration of OPN via SDS− PAGE after TG2 treatment was examined using the Q42A and Q193A mutants. Interestingly, the Q42A mutant exhibited faster migration after TG2 cross-linking, whereas mutation of Gln193 to Ala inhibited the formation of the fast-migrating OPN monomer (Figure 8A). This demonstrates that the altered conformation and electrophoretic mobility of OPNi are caused by TG2-induced intramolecular cross-linking involving Gln193. To examine the specificity of the intramolecular cross-linking reaction mediated by TG2, the Q42A and Q193A mutants were cleaved with thrombin before and after TG2 treatment. In the Q42A mutant, a mass decrease corresponding to formation of an isopeptide bond was observed in only the C-terminal part after TG2 treatment (Figure 8B). The scenario was the opposite in the Q193A mutant, where a single cross-link was observed in the N-terminal part only after TG2 treatment (Figure 8B). Collectively, this shows that the intramolecular cross-linking of OPN catalyzed by TG2 specifically involves Gln42 and Gln193. Comparison of Residues Involved in TG2-Mediated Intramolecular Cross-Linking of Human and Mouse OPN. Residues Lys4, Gln42, Lys154, and Gln193 that are intramolecularly cross-linked in human OPN are conserved in the mouse OPN sequence (Figure S2A). After TG2 treatment, a band corresponding to intramolecular cross-linking was also observed in mouse OPN (Figure S2B). Mouse OPN before and after incubation with TG2 and subsequent digestion with trypsin was analyzed by MALDI-MS. Peptides containing Gln41 (Gln42 in human OPN) were observed only in the trypsin digest of non-cross-linked mouse OPN (Figure S2C). Furthermore, a C-terminal peptide containing Gln178 (Gln193 in human OPN) was absent after TG2 treatment, whereas a peptide with an m/z value at 2929.44 corresponding to a dipeptide cross-linked by the Lys139−Gln178 isopeptide bond (Lys154−Gln193 in human OPN) emerged after TG2 treatment (Figure S2C). Collectively, these results indicate that TG2catalyzed intramolecular cross-linking of mouse and human OPN proceeds in the same way and involves isopeptide bonding between homologous residues. Identification of Cross-Linked OPN in Human Urine. TG2 has previously been shown to be present in urine.22,29 To investigate whether OPNi is present in vivo in urine, OPN purified from human urine (Figure 9A) was digested with trypsin and analyzed by highly sensitive targeted nano-LC− MS/MS using parallel reaction monitoring targeted toward the cross-linked peptide from the C-terminal part of OPNi. A tryptic digest of in vitro-generated OPNi was used as a control in the analysis. A peptide with a mass of m/z 762.7 and a similar chromatographic retention time was observed in both digests. MS/MS fragmentation showed that the detected ion corresponded to the peptides 153SKSK156 and 188AIPVAQDLNAPSDWDSR204 cross-linked via an isopeptide bond (Figure 9B) showing that OPNi exists in vivo.

Figure 8. MALDI-MS and SDS−PAGE analysis of the Q42A and Q193A mutants. (A) The Q42A and Q193A mutants were incubated with TG2 in a 15:1 ratio (w/w) for the indicated times. The reaction products were separated by 18% SDS−PAGE and stained with Coomassie Brilliant Blue. (B) Linear MALDI-MS of thrombin-cleaved Q42A and Q193A mutants untreated and treated with TG2 for 3 h as in panel A. The theoretical molecular masses of the N- and C-terminal parts are 18181.8 and 16843.1 Da, respectively, in Q42A and 18238.9 and 16786.0 Da, respectively, in Q193A.

sample both contained peptides corresponding to Lys14−Glu49 containing one isopeptide bond. Digestion with trypsin or AspN of fraction 3 and 4 suggested that Gln36 or Gln42 was crosslinked to Lys14 in fraction 3 and that Gln36 or Gln42 was crosslinked to Lys19 in fraction 4 (Figure 5B). To investigate whether the cross-links in the N-terminal part of OPN involve Gln36 or Gln42, these residues were mutated to Ala residues. Wild-type OPN and the mutants were subjected to TG2-catalyzed cross-linking, Glu-C digestion, and RP-HPLC as described above. Fractions containing peptides encompassing Lys4, Gln36, and Gln42 were significantly reduced after TG2 treatment of wild-type OPN and the Q36A mutant, but not in the Q42A mutant, as compared to that of untreated wild-type OPN (Figure 6A), showing that Lys4 forms an isopeptide bond with Gln42. Furthermore, the two minor peaks corresponding to an isopeptide bond in the Lys14−Glu49 peptide observed in Figure 5 were also present in the Q36A mutant, and digestion of these fractions with trypsin or endoproteinase Asp-N also indicated that Gln42 can be cross-linked to Lys14 or Lys19 (Figure 6B). The fraction containing a peptide comprising Gln193 were absent after the TG2 treatment of all OPN variants, G

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Figure 9. Identification of cross-linked OPN in human urine. (A) OPN was purified from human urine and separated by SDS−PAGE and stained with Coomassie Brilliant Blue: lane 1, 20 μg; lane 2, 10 μg; lane 3, 5 μg. (B) Urinary OPN and OPNi were digested with trypsin and analyzed by nano-LC−MS/MS. MS/MS spectra of the precursor ion at m/z 762.7 observed in tryptic digests of both OPNi and urinary OPN corresponded to the peptides 153SKSK156 and 188AIPVAQDLNAPSDWDSR204 cross-linked via an isopeptide bond. The MS experiment was repeated three times. Peptides observed in both samples are underlined in the urinary OPN digest.

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DISCUSSION Previous studies of TG2-catalyzed cross-linking of OPN have mainly focused on polymerization of OPN that has been demonstrated using relatively high TG2:OPN ratios [up to 1:1 (w/w)].10−14 In the study presented here, we have used TG2:OPN ratios of 1:100 to 1:20 (w/w) resembling in vivo ratios. At such ratios, TG2 was found to effectively catalyze intramolecular cross-linking of OPN whereas polymerization was not observed. Similar observations have been reported for several other small unstructured proteins such as α-synuclein, Sup35, and τ4RD, which predominantly undergoes intramolecular cross-linking rather than intermolecular polymerization,30,31 upon being incubated with TG2 at physiologically relevant concentrations.25 Intramolecular cross-linking of these proteins has been shown to affect their functionality and is hypothesized to be a defensive mechanism to prevent amyloid formation and protein aggregation in neurodegenerative diseases.25,30 Likewise, intramolecular cross-linking of OPN could be a regulatory mechanism in vivo during, for instance, bone mineralization, wound healing, and urinary stone formation where the TG2:OPN ratio is low.1,15,16,22,32 We have recently shown that virtually all glutamines in both the N- and C-terminal parts of OPN are substrates for TG2.19 In the study presented here, we show that TG2-mediated intramolecular cross-linking of OPN appears to be very specific and involves only a single cross-link in the N- and C-terminal parts of OPN. By site-directed mutagenesis and mass spectrometric analyses, Gln42 and Gln193 were identified as the primary acceptor sites for TG2-catalyzed cross-linking of OPN. Mutation of these residues eliminated the intramolecular cross-linking of OPN, underlining the specificity of the reaction. Interestingly, the Gln residues participating in intramolecular cross-linking of α-synuclein were located in the sequences ValAla-Gln79 and Ala-Pro-Gln109,31 which are similar to the sequences Ala-Pro-Gln42 and Val-Ala-Gln193 containing the reactive glutamines in OPN. However, whether these or similar motifs serve as consensus sequences for TG2 reactive Gln

residues in intramolecular cross-linking remains to be determined. The lysine residues that participate in the formation of isopeptide bonds with Gln42 and Gln193 were also identified. On the basis of the concurrent reduction in absorption at 214 nm of peptides encompassing Lys4 and Gln42 after TG2 treatment (Figures 5 and 6), it seems evident that Gln42 is predominantly linked to Lys4 and to some extent to Lys14 or Lys19. This is in line with our recent study of TG2 reactive residues in human OPN, where Lys4 was observed to be the most TG2 reactive lysine residue in the N-terminal region of OPN 19. Gln193 was observed to participate in multiple cross-links involving Lys154, Lys157 and Lys231. Based on the RP-HPLC trace (Figure 4) the dominating cross-link is that between Lys154 and Gln193. The region containing Lys154, Lys156 and Lys157 has also been observed to be highly TG2 reactive in OPN.19 The observation that the intramolecular cross-linking of OPN was specific regarding the Gln residues and more promiscuous regarding the Lys residues is consistent with the observations of the intramolecular cross-linking of α-synuclein,31 and the general fact that TG2 is much less selective toward amine donor Lys residues than it is with regard to the Gln substrates.33 In a tryptic digest of TG2-treated mouse OPN, an isopeptide bond between residues corresponding to Lys154 and Gln193 in human OPN was identified (Figure S2). Furthermore, tryptic peptides containing residues corresponding to Gln42 were absent after TG2 treatment in mouse OPN. This shows that homologous residues are involved in intramolecular crosslinking of mouse and human OPN. Furthermore, the crosslinking was not affected by the post-translational modifications of OPN, as both recombinant (nonmodified) and phosphorylated and glycosylated human milk OPN were observed to be intramolecularly cross-linked by TG2 (Figure 1). The change in the electrophoretic mobility of OPNi is likely related to the formation of a more compact OPN monomer. Mutation of Gln193, but not Gln42, to Ala eliminated the change in electrophoretic mobility after TG2 treatment. Thus, the cross-link involving Gln193 in the C-terminal part of OPN H

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Biochemistry Funding

seems to introduce the largest structural impact on OPN after TG2 cross-linking. This is consistent with a previous study, showing a significant mobility shift in SDS−PAGE of mouse OPN with a mutation of Gln42 after TG2 treatment.14 TG2-mediated polymerization of OPN has previously been shown to alter the functionality of the protein and especially its integrin binding properties.10,12−14 Interestingly, Gln34 and Gln42 are very well conserved in mammalian OPN sequences, and they constitute the two major TG2 target sites in the Nterminal region of OPN.19 As Gln42 is involved in the intramolecular cross-linking of OPN, it cannot be part of the potential subsequent polymerization, which then likely involves Gln34 because of its high TG2 reactivity. This has also been indicated by site-directed mutagenesis, which suggested that Gln34 or Gln36 were involved in OPN polymerization.14 Furthermore, the C-terminal region of OPN seems to be important for polymerization, as TG2 reactive Lys residues predominantly are located in this region.19 This is consistent with the observations in this study that few TG2 reactive Lys residues in the N-terminus are involved in intramolecular crosslinking. However, whether intramolecular cross-linking is connected with or necessary for subsequent OPN polymerization remains to be determined. The concentrations of TG2 and OPN in human urine are approximately 82 ng/mL22 and 4 μg/mL,23 respectively. Hence, the urinary TG2:OPN ratio is within the range of ratios from 1:100 to 1:20 used in this study where only intramolecular cross-linking of OPN was observed. We therefore tested whether intramolecularly cross-linked OPN was present in human urine and identified two peptides connected via an isopeptide bond between Lys154 and Gln193 indicating that intramolecular cross-linking of OPN does occur in vivo. Urinary OPN was purified using anion chromatography and subsequently RP-HPLC; therefore, the sample is unlikely to contain polymerized OPN, as anion exchange previously has been used to separate in vitro-generated polymers from OPN monomers.11,13 Moreover, no bands corresponding to polymerized OPN were observed in the analyzed urinary OPN preparation (Figure 9A). Several studies have proposed a biological function of TG2mediated intermolecularly cross-linked polymers of OPN.10−14,18 Our data complement these studies and suggest that intramolecular cross-linking of OPN may actually be the major form of TG2-modified OPN in vivo where the TG2:substrate ratio is low.

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This work was supported by The Danish Council for Independent Research-Natural Sciences (Grant 12-126168). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS OPN, osteopontin; TG2, transglutaminase 2; OPNi, intramolecularly cross-linked osteopontin; RP, reversed phase; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01153. TG2-mediated cross-linking of OPN (Figure S1) and TG2-mediated intramolecular cross-linking of mouse OPN (Figure S2) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (45) 87155461. Author Contributions

B.C. and E.D.Z. contributed equally to this work. I

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